PROJECT SUMMARY/ABSTRACT This application is a renewal of an ongoing project in which four investigators with different but complementary expertise have worked together to understand how the T cell antigen receptor (TCR) regulates the proximal tyrosine kinases (SFKs, Syk kinases and Tec kinases) that control critical downstream tyrosine phosphorylation. We have made considerable progress in understanding the structural basis for the specificity differences that are encoded in the kinase domains and the autoregulatory constraints that control the activities of these kinases, but a full understanding will require new approaches. We propose to capitalize on our current progress and bring together approaches from structural biology, physical sciences, proteomics, immunology, and computational biology to perform studies that are aimed at understanding the distinct features of the T cell-expressed SFKs, Syk and Tec kinases that make the individual kinases more suitable for antigen receptor signaling in T cells than in B cells. We hypothesize that the characteristics of Lck and Fyn, ZAP-70 and Itk and their signaling regulators have been optimized in T cells to establish signaling circuitry that serves to maintain a basal signaling state that is resistant to perturbations by non- agonist peptides and also establishes a sensitive threshold for optimal recognition and response to agonist pMHC. We will explore this hypothesis in experiments designed to: 1) understand the unique features of the proximal kinases that are advantageous in TCR signaling; 2) define the regulatory mechanisms that constrain the activity of the proximal tyrosine kinases; 3) determine how TCRs maintain basal homeostasis and distinguish biological noise from antigenic stimuli; and, 4) define the key intracellular events needed to initiate downstream signal propagation by the TCR.